Receptor tyrosine kinases (RTKs) are involved in a broad spectrum of cell growth and differentiation events. RTKs are classified based on sequence homology and domain organization. Type I RTKs include the epithelial growth factor receptor (EGFR) and the Human EGF Receptor homologues HER2 (HER2/neu, p185), HER3 and HER4 (also named c-erbB1-4). Overexpression of several members of this receptor family, especially EGFR and HER2, is associated with a variety of solid tumor malignancies (see, e.g. Dougall et al. (1993) J Cell Biochem 53, 61-73; Berchuck et al. (1990) Cancer Res 50, 4087-91; Schneider et al. (1989) Cancer Res 49, 4968-71; Yokota et al. (1988) Oncogene 2, 283-7; and Slamon et al. (1989) Science 244, 707-12). Overexpression of HER2 is found in 20-30% of breast cancers and results in ligand independent activation and more aggressive growth behavior (see, e.g. Slamon et al. (1989) Science 244,707-12).
Among the four mammalian type I RTKs, HER3 is unique because of its catalytically deficient kinase domain (see, e.g. Guy et al. (1994) Proc Natl Acad Sci USA 91, 8132-6), its high propensity to self-associate in the absence of ligand (see, e.g. Landgraf et al. (2000) Biochemistry 39, 8503-8511) and the ability of the monomeric species of HER3ECD to assume a locked conformation, using an intramolecular tether (see, e.g. Cho et al. (2002) Science 297, 1330-3). HER3 binds a variety of isoforms of the EGF homolog heregulin, and signaling relies on heterodimerization with other RTKs, preferentially HER2 (see, e.g. Sliwkowski et al. (1994) Journal of Biological Chemistry 269, 14661-5). HER2 has a potent cytoplasmic kinase domain but is deficient in ligand binding. Simultaneous overexpression of both HER2 and HER3 is found in several cancers (see, e.g. see, e.g. Naidu et al. (1998) Br J Cancer 78, 1385-90; and Krahn et al. (2001) Eur J Cancer 37, 251-9), and the increased drug resistance in many HER2 overexpressing cancers depends on increased levels of HER3 or EGFR (see, e.g. Chen et al. (2000) Biochem Biophys Res Commun 277, 757-63).
Ligand controlled signaling by type I RTKs involves receptor dimers. However, at elevated expression levels HER2 and other RTKs are likely to be engaged in a broader range of interactions. Activation of HER2 has been shown to result in the formation of large clusters of activated receptors from preexisting smaller clusters (see, e.g. Nagy et al. (1999) J Cell Sci 112 (Pt 11), 1733-41). For EGFR, ligand-independent interactions of receptors have been implicated in the rapid spread of signal over the entire surface of the cell after localized stimulation with immobilized ligand (see, e.g. Verveer et al. (2000) Science 290, 1567-70).
The extracellular domains of RTKs (ECDs) provide attractive targets for macromolecular anti-cancer drugs. Examples include soluble ECDs of the receptors (see, e.g. Azios et al. (2001) Oncogene 20, 5199-209) and antibodies against the ECDs (see, e.g. Ranson et al. (2002) Oncology 63 Suppl 1, 17-24; and Agus et al. (2002) Cancer Cell 2, 127-37). Herceptin, a humanized antibody against HER2, has shown great promise in the treatment of HER2 overexpressing breast cancers (see, e.g. Pegram et al. (1999) Oncogene 18, 2241-51), thus demonstrating two important points. First, interference by large macromolecules with this first level of the signaling cascade holds therapeutic potential. Second, intrinsic toxicity is not required for a drug to be effective against cells that overexpress growth factor receptors.
As macromolecular drugs, RNA aptamers against RTKs have advantages over proteins. Libraries of randomized RNAs can be generated in vitro with a very high level of sequence complexity. Libraries can be screened in vitro using SELEX (Systematic Evolution of Ligands by EXponential enrichment) (see, e.g. Gold et al. (1995) Annu Rev Biochem 64, 763-97). A variety of chemical modifications exists for nucleic acids, such as the incorporation of radiolabels, fluorescent probes, or cross-linking reagents, and modifications to the backbone or specific bases can be introduced at will, thereby adding functionality and stability. RNA aptamers are non-immunogenic, and the use of fluorine or amino groups in the 2′ position significantly enhances the half-life of RNA-aptamers in serum.
In recent years, aptamers have been selected successfully against several extracellular protein ligands, such as TGFβ, PDGF, basic FGF and VEGF (see, e.g. Golden et al. (2000) J Biotechnol 81, 167-78; Pietras et al. (2001) Cancer Res 61, 2929-34; Jellinek et al. (1995) Biochemistry 34, 11363-72; and Jellinek et al. (1994) Biochemistry 33, 10450-6). Aptamers against VEGF shrink tumors in mice and have shown promise for the treatment of macular dysfunction (see, e.g. Martin et al. (2002) Retina 22, 143-52; and Kim et al. (2002) Proc Natl Acad Sci USA 99, 11399-404). An aptamer against the proinflammatory cytokine oncostatin M is being evaluated for use against rheumatoid arthritis (see, e.g. Rhodes et al. (2000) J Biol Chem 275, 28555-61), and aptamers against blood coagulation factors VIIa and IXa are under investigation as anticoagulants (see, e.g. Rusconi et al. (2000) Thromb Haemost 84, 841-8; and Rusconi et al. (2002) Nature 419, 90-4).
As a target for aptamer selection, RTKs stand out through their large size. The extracellular domains of type I RTKs are heavily glycosylated, may form several higher molecular weight complexes, and a variety of distinct conformations are likely to exist. These differences pose a considerable challenge for the application of SELEX to RTKs. HER3 exemplifies these challenges, because of its high propensity to self-associate. Consequently, there is a need in the art for methods that allow the identification aptamers to RTKs such as HER3 as well as specific aptamers that recognize these molecules. The invention disclosed herein satisfies this need.